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Computational Structural Analysis and Kinetic Studies of a Cytosolic Glutamine Synthetase from Camellia sinensis (L.) O. Kuntze

The Protein Journal volume 28, pages 428–434 (2009)Cite this article

Abstract

Structural analysis of a cytosolic glutamine synthetase from Camellia sinensis (CsGS) has been conducted employing computational techniques. This was conducted to compare its structural aspects with other known structures of GS. The disordered residues and their distribution in CsGS are in close comparison to earlier reported GS. The 3-D structure of CsGS also showed high degree of similarity with the only known crystal structure of GS from Zea mays. The K m values observed with recombinant CsGS for all the three substrates are higher compared to rice, Arabidopsis, maize and human. This suggests lower affinity of CsGS for substrates. Further, kinetic mechanism of CsGS catalysis was investigated using initial velocity analysis and product inhibition studies. Initial velocity data eliminate the possibility of ping-pong mechanism and favor the random mechanism of catalysis. Through product inhibition studies, ADP was found to be a competitive inhibitor with respect to ATP and noncompetitive inhibitor versus both glutamate and ammonium. While, glutamine and inorganic phosphate were found to be non-competitive inhibitors of ATP, glutamate and ammonia. Taken together, these observations are consistent with a random catalysis mechanism for the CsGS where the binding order of certain substrates is kinetically preferred by the enzyme.

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Scheme 1
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Abbreviations

A620 :

Absorbance at 620 nm

ADP:

Adenosine diphosphate

ATP:

Adenosine triphosphate

NH3 :

Ammonia

CsGS:

Camellia sinensis glutamine synthetase

EDTA:

Ethylenediaminetetraacetic acid

GS:

Glutamine synthetase

GOGAT:

Glutamine oxoglutarate aminotransferase

Pi:

Inorganic phosphate

UDP:

Uridine diphosphate

ZmGS:

Zea mays glutamine synthetase

References

  1. Almassy RJ, Janson CA, Hamlin R, Xuong N, Eisenberg D (1986) Nature 323:304–309

    Article  CAS  Google Scholar 

  2. Anderson MS, Eveland SS, Onishi HR, Pompliano DL (1996) Biochemistry 35:16264–16269

    Article  CAS  Google Scholar 

  3. Arnold K, Bordoli L, Kopp J, Schwede T (2006) Bioinformatics 22:95–201

    Google Scholar 

  4. Bernard SM, Møller AL, Dionisio G, Kichey T, Jahn TP, Dubois F, Baudo M, Lopes MS, Tercé-Laforgue T, Foyer CH, Parry MA, Forde BG, Araus JL, Hirel B, Schjoerring JK, Habash DZ (2008) Plant Mol Biol 67:89–105

    Article  CAS  Google Scholar 

  5. Bhandari B, Roesler WJ, DeLisio KD, Klemm DJ, Ross NS, Miller RE (1991) J Biol Chem 266:7784–7792

    CAS  Google Scholar 

  6. Brekken DL, Phillips MA (1998) J Biol Chem 273:26317–26322

    Article  CAS  Google Scholar 

  7. Brugie`re N, Dubois F, Limami A, Lelandais M, Roux Y, Sangwan R, Hirel B (1999) Plant Cell 11:1995–2011

    Article  CAS  Google Scholar 

  8. Canovas F, Avila C, Canton FR, Canas R, de la Torre F (2007) J Exp Bot 58:2307–2318

    Article  CAS  Google Scholar 

  9. Chiurazzi M, Meza R, Lara M, Lahm A, Defez R, Iaccarino M, Espín G (1992) Gene 119:1–8

    Article  CAS  Google Scholar 

  10. Cooper BF, Rudolph FB (1995) Methods Enzymol 249:188–211

    Article  CAS  Google Scholar 

  11. de la Torre F, García-Gutiérrez A, Crespillo R, Cantón FR, Avila C, Cánovas FM (2002) Plant Cell Physiol 43:802–809

    Article  Google Scholar 

  12. Dubois F, Brugière N, Sangwan RS, Hirel B (1996) Plant Mol Biol 31:803–817

    Article  CAS  Google Scholar 

  13. Eisenberg D, Gill HS, Pfuegl GMU, Rotstein SH (2000) Biochim Biophys Acta 1477:122–145

    CAS  Google Scholar 

  14. Emanuele JJ, Jin H, Yanchunas J, Villafranca JJ (1997) Biochemistry 36:7264–7271

    Article  CAS  Google Scholar 

  15. Filser DM, Moscatelli C, Lamberti A, Vincze E, Guida M, Salzano G, Iaccarino M (1986) J Gen Microbiol 132:2561–2569

    CAS  Google Scholar 

  16. Finnemann J, Schjoerring JK (2000) Plant J 24:171–181

    Article  CAS  Google Scholar 

  17. Geourjon C, Deléage G (1995) Comp Appl Biosc 11:681–684

    CAS  Google Scholar 

  18. Gill HS, Pfluegl GM, Eisenberg D (1999) Acta Crystallogr D Biol Crystallogr 55:865–868

    Article  CAS  Google Scholar 

  19. Habash DZ, Massiah AJ, Rong HL, Wallsgrove RM, Leigh RA (2001) Ann Appl Biol 138:83–89

    Article  CAS  Google Scholar 

  20. Hirel B, Gadal P (1980) Plant Physiol 66:619–623

    Article  CAS  Google Scholar 

  21. Hirel B, Lea PJ (2001) Ammonia assimilation. In: Lea PJ, Morot-Gaudry J-F (eds) Plant nitrogen. Springer-Verlag, Berlin, pp 79–99

    Google Scholar 

  22. Ishiyama K, Inoue E, Watanabe-Takahashi A, Obara M, Yamaya T, Takahashi H (2004) J Biol Chem 279:16598–16605

    Article  CAS  Google Scholar 

  23. Jez JM, Cahoon RE, Chen S (2004) J Biol Chem 279:33463–33470

    Article  CAS  Google Scholar 

  24. Joyce BK, Himes RH (1966) J Biol Chem 241:5725–5731

    CAS  Google Scholar 

  25. Kamachi K, Yamaya T, Mae T, Ojima K (1991) Plant Physiol 96:411–417

    Article  CAS  Google Scholar 

  26. Kichey T, Le Gouis J, Sangwan B, Hirel B, Dubois F (2005) Plant Cell Physiol 46:964–974

    Article  CAS  Google Scholar 

  27. Krajewski WW, Collins R, Holmberg-Schiavone L, Jones TA, Karlberg T, Mowbray SL (2008) J Mol Biol 375:217–228

    Article  CAS  Google Scholar 

  28. Krajewski WW, Jones TA, Mowbray SL (2005) Proc Natl Acad Sci USA 102:10499–10504

    Article  CAS  Google Scholar 

  29. Lam HM, Coschigano KT, Oliveira IC, Melo-Oliveira R, Coruzzi GM (1996) Ann Rev Plant Physiol Plant Mol Biol 47:569–593

    Article  CAS  Google Scholar 

  30. Liaw SH, Pan C, Eisenberg D (1993) Proc Natl Acad Sci USA 90:4996–5000

    Article  CAS  Google Scholar 

  31. Listrom CD, Morizono H, Rajagopal BS, McCann MT, Tuchman M, Allewell NM (1997) Biochem J 328:159–163

    CAS  Google Scholar 

  32. Man HM, Boriel R, El-Khatib R, Kirby EG (2005) New Phytol 167:31–39

    Article  CAS  Google Scholar 

  33. Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, Dubois F, Balliau T, Valot B, Davanture M, Terce′-Laforgue T, Quillere′ I, Coque M, Gallais A, Gonzalez-Moro MB, Bethencourt L, Habash DZ, Lea PJ, Charcosset A, Perez P, Murigneux A, Sakakibara H, Edwards KJ, Hirel B (2006) Plant Cell 18:3252–3274

    Article  CAS  Google Scholar 

  34. Masclaux C, Quillere I, Gallais A, Hirel B (2001) Ann Appl Biol 138:69–81

    Article  CAS  Google Scholar 

  35. Meek TD, Villafranca JJ (1980) Biochemistry 19:5513–5519

    Article  CAS  Google Scholar 

  36. Meierjohann S, Walter RD, Muller S (2002) Biochem J 363:833–838

    Article  CAS  Google Scholar 

  37. Miflin BJ, Lea PJ (1980) In: Miflin BJ (ed) The biochemistry of plants. Academic Press, New York, pp 169–202

  38. Mullins LS, Zawadzke LE, Walsh CT, Raushel FM (1990) J Biol Chem 265:8993–8998

    CAS  Google Scholar 

  39. Pellegrini M, GrÖnbech-Jensen N, Kelly JA, Pfuegl GMU, Yeates TO (1997) Protein Struct Funct Genet 29:426–432

    Article  CAS  Google Scholar 

  40. Pereira S, Pissara J, Sunkel C, Salema R (1995) Ann Bot 77:429–432

    Article  Google Scholar 

  41. Rana NK, Mohanpuria P, Kumar V, Yadav SK (2009) Mol Biol Rep. doi: 10.1007/s11033-009-9559-6

  42. Rana NK, Mohanpuria P, Yadav SK (2008) Biol Plant 52:361–364

    Article  CAS  Google Scholar 

  43. Rana NK, Mohanpuria P, Yadav SK (2008) Mol Biotechnol 39:49–56

    Article  CAS  Google Scholar 

  44. Sakakibara H, Shimizu H, Hase T, Yamazaki Y, Takao T, Shimonishi Y, Sugiyama T (1996) J Biol Chem 271:29561–29568

    Article  CAS  Google Scholar 

  45. Sakurai N, Hayakawa T, Nakamura T, Yamaya T (1996) Planta 200:306–311

    Article  CAS  Google Scholar 

  46. Segel IH (1975) Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. Wiley, New York

    Google Scholar 

  47. Tabuchi M, Sugiyama K, Ishiyama K, Inoue E, Sato T, Takahashi H, Yamaya T (2005) Plant J 42:641–651

    Article  CAS  Google Scholar 

  48. Unno H, Uchida T, Sugawara H, Kurisu G, Sugiyama T, Yamaya T, Sakakibara H, Hase T, Kusunoki M (2006) J Biol Chem 281:29287–29296

    Article  CAS  Google Scholar 

  49. Usdin KP, Zappe H, Jones DT, Woods DR (1986) Appl Environ Microbiol 52:413–419

    CAS  Google Scholar 

  50. Valentine RC, Shapiro BM, Stadtman ER (1968) Biochemistry 7:2143–2152

    Article  CAS  Google Scholar 

  51. Viola RE, Cleland WW (1982) Methods Enzymol 87:353–366

    Article  CAS  Google Scholar 

  52. Yamashita MM, Almassy RJ, Janson CA, Cascio D, Eisenberg D (1989) J Biol Chem 264:17681–17690

    CAS  Google Scholar 

  53. Yao J, Patrone JD, Dotson GD (2009) Biochemistry 48:2799–2806

    Article  CAS  Google Scholar 

Download references

Acknowledgments

I would like to acknowledge Dr. P. S. Ahuja, Director, IHBT for his continuous support and guidance to conduct this work. The financial assistance from Council of Scientific and Industrial Research (CSIR) and Department of Science and Technology (DST), Govt. of India is duly acknowledged.

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  1. Biotechnology Division, Institute of Himalayan Bioresource Technology, CSIR, Palampur, 176061, India

    Sudesh Kumar Yadav

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Yadav, S.K. Computational Structural Analysis and Kinetic Studies of a Cytosolic Glutamine Synthetase from Camellia sinensis (L.) O. Kuntze. Protein J 28, 428–434 (2009). https://doi.org/10.1007/s10930-009-9210-3

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